This study investigated the effects of the putative BK channel opener VSN16R on neuronal excitability of CA1 pyramidal neurons. We demonstrated for the first time in native mammalian neurons that VSN16R increased fAHP, decreased ISI and shortened AP width, effects consistent with increased BK_Ca activation. VSN16R actions were dependent on stimulus protocols used to induce neuronal firing, such that VSN16R effects, when applied alone, were seen at higher, but not lower, frequency stimulations. Such effects of VSN16R were blocked by IBTX, with IBTX itself having effects on the AP waveform. The latter data are consistent with previous reports of IBTX action at BK_Ca channel activity in CA1 neurons [8,11]. This study extends recent demonstrations that VSN16R increased single channel activity of large conductance BK_Ca channels in an excised inside-out patch from a human endothelial cell line or in human cerebral cortex neural HCN-2 cell lines [2] to a native neuronal preparation. This study further confirmed that cannabinoid-like compounds include BK_Ca channels amongst their repertoire of molecular targets.

BK_Ca channels are widely reported to show three major biophysical phenotypes, dependent on expression of BK_Ca α subunits with different accessory β subunits; thus, inactivating BK_Ca channels are composed of α + β2, non-inactivating type I channels of α alone and type II channels of α + β4 (reviewed in [12]). Related to this, β2 and β4 are the predominant BK_Ca β subunits expressed in the CNS [13], and it has been proposed that β2 expression is associated with IBTX sensitivity, while β4 expression confers IBTX resistance [14,15,16]. With further relevance here, original studies have shown that VSN16R blocked BK_Ca channels in Ea.Hy926 cells, which predominantly express α + β4 subunits, but had no action at BK_Ca channels in porcine aorta endothelium, which reportedly lack all β subunits [2]. CA1 neurons express inactivating, IBTX-sensitive BK_Ca channels [8,17]. The present data demonstrate that VSN16R actions in CA1 neurons are sensitive to IBTX block, in agreement with data in rat mesenteric arteries, where VSN16R induced vasorelaxation in an endothelium-dependent and IBTX-sensitive manner [2]. Together, these studies argue against a totally β4-selective activity for VSN16R. This argument is supported further by the first use of 7-Pra-MarTx, a toxin modified at the Lys7 residue of martentoxin. Martentoxin is a 37 amino acid peptide purified from scorpion (Buthus martensi Karsch) venom, which has been proposed as a toxin with selectivity for BK_Ca channels composed of α + β4, over those composed of the α subunit alone [10,18]. In the Shi et al. study [10], martentoxin inhibition was shown to be lost under conditions of increased cytoplasmic Ca²⁺. However, martentoxin also blocks BK_Ca in adrenal chromaffin cells [9] which may express α + β2 subunits. Conversely, martentoxin can enhance glioma BK_Ca currents and those composed of α + β1 subunits in an expression system [19]; martentoxin also acted in a Ca^2+--dependent fashion in this study, such that increased cytoplasmic Ca²⁺ promoted current potentiation. These data suggest that specific toxins, such as martentoxin and their analogues, may be useful pharmacological agents to discriminate BK_Ca subtypes, but that intracellular Ca²⁺ dynamics, widely known to modulate BK_Ca activity, can also modulate toxin effects (see also [18]). 7-Pra-MarTx, at least partially, co-localises with β4 subunits in Ea.Hy926 cells (Darryl Overby and Jacques Bertrand, Imperial College London, personal communication). Here, 7-Pra-MarTx had no major effects on fAHP or ISI, but did have some actions, causing a shortening of AP width; of note here is that 7-Pra-MarTx effects on AP width occurred in the opposite direction to those of the proposed β2-subunit-preferring IBTX. Given that CA1 neurons are reported to express β4 subunits, albeit at lower levels than the prominent β4 expression reported in other hippocampal neurons such as dentate gyrus and CA3 pyramidal neurons [20,21,22], the lack of effect of 7-Pra-MarTx on VSN16R-mediated effects is also consistent with VSN16R not being a β4 selective agent.

Of further interest here was that VSN16R had apparent addition effects in the presence of 7-Pra-MarTx. Thus, VSN16R actions under low frequency stimulation protocol were uncovered; here, VSN16R-mediated increased fAHP and decreased first ISI (as seen for VSN16R when applied alone under high, but not low, frequency stimulation). These actions are predicted to reduce neuronal hyperexcitability. A putative explanation of these data is that 7-Pra-MarTx block of β4 subunits, which are regarded as inhibitory subunits [12], further reveals VSN16R action at β2 (and/or α) channels that coexist in CA1 neurons. In this regard, different BK_Ca channel subtypes are reported to coexist in different neurons, including mossy fiber presynaptic terminals [23] and Purkinje cells [24], and BK_Ca ligand sensitivity is often associated with subpopulations of given neurons, for example, as shown for IBTX sensitivity in dentate gyrus neurons [20,25]. On the basis of widespread putative IBTX-resistant β4 subunit expression, but comparatively rarer examples of IBTX-resistant channels, it has also been proposed that α subunits can be less than fully saturated with β4, and, thus, that IBTX can still block such channels [12]. It is also possible that BK_Ca diversity may extend to mixed accessory subunits, such as β2 and β4 heteromultimers.

BK_Ca channels are key contributors to neuronal excitability, and it may be predicted that BK_Ca opening would cause general hyperpolarization and thereby reduce neuronal hyperexcitability, as proposed for VSN16R effects in spasticity [2]. Such actions may prevent epileptiform discharges and be useful agents for seizure control. For example, BK_Ca loss-of-function has been implicated in conditions such as temporal lobe epilepsy [26]. Our data demonstrate that VSN16R increases and IBTX decreases firing frequency in CA1 neurons. Although this may appear counter-intuitive, BK_Ca channels have been shown to facilitate high frequency firing of CA1 neurons via effects on rapid spike repolarization and the fast afterhyperpolarization, which ultimately reduce Na⁺ channel inactivation and activate slower, delayed rectifier K⁺ channels [8]. In general, for BK_Ca channels it appears that a correct balance of excitability must be maintained, as both loss-of-function and gain-of-function BK_Ca mutations have been associated with different forms of epilepsy; correspondingly, pharmacological intervention by BK_Ca openers and inhibitors have reported utility in preventing epileptiform discharges in different models (see [27,28,29]. Overall, increasing our pharmacological knowledge of BK_Ca channel activators and inhibitors and their role(s) in modulating different forms of epilepsy, and other diseases of hyperexcitability, and of how different BK heteromonomers and/or heteromultimers may be targeted selectively, is a key aim. Here, we provide further evidence that the cannabinoid-like VSN16R compound, which has demonstrated good CNS penetration, may represent a useful starting point for therapeutic development of BK_Ca openers.